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Pre-infection Post-infection 5 BMB400, 4_4_04 Visible effects on DNA during viral infection T4 phage DNA Phage cannot replicate on their own – Infect a host cell Once inside the host cell, they either remain quiescent (as prophage) or use the cell’s replication machinery to produce many copies of themselves – This replication may or may not lead to destruction of the host cell (lytic) 6 3

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BMB400, 4_4_ Why are phage important? – They cause diseases – They are useful tools in molecular biology Phage are often used as “replacement vectors” - Part of their DNA is removed and replaced with other DNA of interest (e.g., human DNA) - This recombinant DNA is repackaged into phage; phage infect host bacteria - Phage use bacteria to replicate, therefore replicating the DNA of interest Bacteriophage 8  bacteriophage as a replacement vector

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9 toxin prophage Corynebacterium diptheriaea Phage produces diptheria toxin This is what makes people sick insertion site C.diptheriaea without phage strain produces no toxin Does not cause diptheria BMB400, 4_4_04 Phage conversion Dormant prophage – integrated bacteriophage – carries genes that alter the phenotype of the microbe - best examples are pathogens and toxin production Lysis or Lysogeny Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium (bacteriophage T4) Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively – No progeny – No lysis of the host Bacteriophage lambda can do either. 10 5

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7 13 BMB400, 4_4_04 Elements of lysogeny The phage genome integrated into the host bacterial genome is a prophage. Bacterium carrying the prophage is a lysogen. Lysogens are immune to further infection by similar phage because the phage functions are repressed in trans. Induction of the lysogen leads to excision of the prophage, replication of the phage DNA, and lysis of the host bacterium. Site-specific recombination in bacteriophage. X-Ray structure of integration host factor (IHF) in complex with a 35-bp target DNA. 14

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BMB400, 4_4_ Early translation – products are usually enzymes to shut down host and synthesize viral nucleic acids Late transcription/translation produces structural proteins The Bacteriophage T4 16 Lytic virus T4 has linear DNA with specific hydroxymethylcytosine, instead of cytosine, resistent to endonuclease of the host Produce enzyme in DNA replication that similar to those host enzyme in large amount Encode three group protein (early, middle and late proteins)

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BMB400, 4_4_ A genetic map of bacteriophage. 18 The Bacteriophage lambdha Lytic and lysogenic virus Genome: double strand DNA, has cohesive ends (cos) Early transcription begin on PL and PR to give the product of N and Cro protein (regulator) N antiterminator (produce O, P (induce replication), Cll and Clll The antiterminator is not effective before termination before the Q gene (little Q protein is produced, Q is antiterminator to transcribe the late genes)) Cro regulator between lytic dand lysogenic pathways by blocking PL and PR (binding OL and OR (site 1, 2, 3) CII (aktivator protein by activate PE to express CI and integrase) and CIII is stabilization of CII.